Persistent antigen at vaccination sites induces tumor-specific CD8⁺ T cell sequestration, dysfunction and deletion

Abstract

To understand why cancer vaccine-induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund's adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8(+) T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

Figure 1

Vaccination with gp100 in IFA induces CD8⁺ T cell priming followed by hyporesponsiveness. (a) Mice received gp100-specific CD90.1⁺ pmel-1 T cells and gp100/IFA s.c. or saline/IFA control vaccination or were left unvaccinated. On day 42, mice were boosted with VSV.gp100 or control VSV.OVA. Levels of CD90.1⁺ pmel-1 T cells in blood (mean ± s.e.m.) are shown. (b) Mice received pmel-1 T cells and VSV.gp100 i.v. on day 0 and/or s.c. vaccination with gp100/IFA on day 0 or 31. Levels of pmel-1 T cells in blood are shown. (c) Mice were vaccinated with s.c. OVA/IFA or saline/IFA and boosted on day 36 with VSV.OVA. OVA tetramer⁺ endogenous CD8⁺ T cells in blood are shown. (d) Albino C57BL/6 mice bearing 7 d, palpable, B16 melanoma were vaccinated with gp100/IFA s.c. and/or VSV.gp100 i.v. and received 1 × 10⁶ v-effLuc-transduced pmel-1 T cells i.v., followed by 3 d of IL-2. Mean number of pmel-1 T cells in blood (top) and representative imaged mice 4 d after vaccination (bottom) photons s⁻¹ are shown. T, tumor; V, gp100/IFA vaccination site; S, spleen. (e) Albino C57BL/6 mice bearing 7 d, palpable, B16 melanomas were vaccinated with gp100/IFA or saline/IFA s.c. and received v-effLuc-transduced pmel-1 T cells i.v. and 3 d of IL-2. Pmel-1 T cells were visualized on day 7. Circles indicate location of s.c. B16 tumor. Color bars represent photons s^−1. Data shown are representative of at least two experiments, each with (n=5 per group per experiment). * P<0.05.

Figure 2

Vaccination with gp100/IFA induces chronic antigen presentation and T cell sequestration. (a) Albino C57BL/6 mice bearing 7 d, palpable s.c. B16 tumors were vaccinated s.c. with gp100/IFA on day 0 or −30 or saline/IFA on day 0 and received 1 × 10⁶ v-effLuc-transduced pmel-1 T cells i.v. followed by 3 d of IL-2. After 4 d, mice were imaged for pmel-1 T cell localization, a representative mouse (photons s⁻¹) is shown. Kinetics of absolute pmel-1 T cell luminescence (mean ± s.e.m., n=5) in different organs/tissues after day 0 vaccination are plotted (b) Mice were vaccinated s.c. with gp100/IFA or saline/IFA. After 30 d, vaccine-draining lymph nodes (VdLN), spleen and the vaccine depot itself were removed and VdLN cells, splenocytes, recovered vaccine emulsion or 1 μM of gp100 peptide were added to cultured pmel-1 effector T cells; 4 h intracellular IFN-γ staining in pmel-1 T cells (mean ± s.e.m.) is shown (c) Mice were vaccinated with gp100/IFA on day 0, followed by pmel-1 T cells on day 0 or on day 96. Levels of pmel-1 T cells (mean ± s.e.m.) in blood are shown. Data shown are representative of at least two experiments, each with (n=5 per group per experiment). * P<0.05

Figure 3

Vaccination-induced CD8+ T cell apoptosis at the vaccination site is driven by antigen and IFN-γ and requires host FasL. (a) Mice received pmel-1 T cells and gp100/IFA s.c. After 48 h, 1000 primed CD44⁺ CD8⁺ T cells were sorted from VdLN and transferred i.v. to recipient mice bearing 48 h old s.c. gp100/IFA or saline/IFA vaccine depots. Twenty-eight d later, all recipient mice were boosted with VSV.gp100; subsequent levels of pmel-1 T cells in blood are shown. (b) Mice received CD90.1⁺ pmel-1 T cells and gp100/IFA and saline/IFA vaccination on opposite flanks. 6 d later, apoptotic death of CD90.1⁺ pmel-1 T cells from VdLN was quantified (mean ± s.e.m., n=10). Absolute number of pmel-1 T cell (mean ± s.e.m., n=5) per VdLN is plotted (right panel). (c) Fas expression on pmel-1 T cells 6 d after gp100/IFA vaccination. (d) C57BL/6 and FasL-KO mice received pmel-1 T cells and gp100/IFA vaccination, 6 d later apoptotic cell death of pmel-1 T cells at the vaccination site was measured by flow cytometry. (e) C57BL/6 and IFN-γ-KO mice received OVA/IFA s.c., 6 d later Fas expression and (f) apoptotic cell death was measured by gating on endogenous OVA tetramer⁺CD8⁺ T cells recovered from the vaccination site; absolute number of CD8⁺ OVA tetramer⁺ T cells at vaccination site is also shown. Data are representative of at least two experiments, each with 5 mice per group. * P <0.05

Figure 4

T cell sequestration and deletion is overcome by vaccination with a short-lived, water-based vaccine formulation (a) Mice were vaccinated s.c. with gp100/IFA or saline/IFA. 1 × 10⁵ freshly isolated VdLN cells or splenocytes or 1 μM of gp100 peptide were added to 1 × 10⁵ cultured pmel-1 effector T cells 6 d later. 4 h intracellular IFN-γ staining in pmel-1 T cells is shown. (b) Mice received pmel-1 T cells and gp100/IFA or gp100/saline vaccine s.c. and 50 μg anti-CD40 mAb s.c. and were boosted after 31 d with VSV.gp100 i.v; pmel-1 T cells levels (mean ± s.e.m.) in blood are shown. (c) Mice received pmel-1 T cells and gp100/IFA or gp100/saline vaccine s.c. and covax (50 μg anti-CD40 mAb s.c., 50 μg imiquimod cream applied on the vaccination site and 3 d of 100,000 IU rhIL-2 i.p.) on d 0 and again but without imiquimod on d 21; pmel-1 T cells levels in blood are shown. (d) Mice (n=5) were treated as in (c) and apoptotic cell death in VdLN was measured 6 d later. (e) Mice (n=5) were treated as in (c) and 5 d later expression of Bcl-2, Bcl-xL and CD127 in pmel-1 T cells from VdLN was measured by flow cytometry. (f) Mice (n=10) received pmel-1 T cells and gp100/IFA or gp100/saline vaccine s.c. and covax on day 0 and B16 tumor challenge on day 41. Shown are levels of pmel-1 T cells in blood (left panel) and tumor size for individual mice (right panels). (g) Mice (n=10) bearing 7 d, palpable s.c. B16 melanoma received pmel-1 T cells and s.c. vaccination with gp100/saline or gp100/IFA and covax on day 0. Shown are pmel-1 T cell levels in blood (left panel) and tumor size in individual mice (right panels). Data shown are representative of at least two experiments, each with 5 mice per group. * P <0.05, NS (not significant): P>0.05.

Figure 5

Vaccination with a short-lived, water-based vaccine formulation allows T cell localization to tumors (a) Albino C57BL/6 mice bearing 7 d palpable B16.white melanoma received v-effLuc-transduced pmel-1 effector T cells i.v. and gp100/IFA+covax or gp100/saline+covax. After 7 d, pmel-1 T cells were visualized (top left panels) and quantified in tumor and vaccination site. Absolute pmel-1 T cell luminescence (mean ± s.e.m., n=5) in tumor and ratio of pmel-1 T cell luminescence in tumor vs. vaccination site (mean ± s.e.m., n=5) are plotted (right panels). The graph is representative of three replicate experiments. Vaccination sites of C57BL/6 mice that received pmel-1 and gp100/IFA+covax or gp100/saline+covax s.c. were photographed 31 d after vaccination (bottom left panels). (b) Mice bearing 7 d palpable B16 melanoma received pmel-1 T cells and gp100/IFA or gp100/saline saline/IFA vaccine s.c. and covax and 6 d later tumor and vaccination sites were excised and levels of pmel-1 T cell infiltration were quantified by flow cytometry. (c) CXCR3 expression by pmel-1 T cells in PBL 6 d after vaccination (d) Supernatant from tumor and vaccination site homogenates from (b) were used to measure cytokine/chemokine concentrations (mean ± s.e.m.). Data shown are representative of three experiments, each with 5 mice per group. * P <0.05, NS (not significant): P>0.05.

Figure 6

pmel-1 T cell phenotype after vaccination with gp100/IFA + covax vs. gp100/saline + covax. (a) Mice bearing 7 d palpable B16 melanoma received pmel-1 T cells and gp100/IFA or gp100/saline saline/IFA vaccine s.c. and covax. Gene expression profiles were measured 6 and 21 d later. (b) Expression of indicated molecules in peripheral blood was assessed over time by flow cytometry by gating on CD8⁺CD90.1⁺ pmel-1 T cells. (c) Proliferation, expression of inhibitory markers and cytokine production in pmel-1 T cells from VdLN 23 d after vaccination with gp100/IFA vs. gp100/saline. * P <0.05